Detection And Function Study Of Nicotinic Acid Receptor On Murine Islet β-cells

In this study, in order to understand the mechanism responsible for the hyperglycemic effect, we investigated the expression of nicotinic acid receptor PUMA-G and its signaling in murine islet beta cells.The results were summarized as below:Firstly, total RNA from murine islets were used for quantification of PUMA-G expression by real time PCR. RNA samples without reverse transcription were used as negative controls. The results showed that the transcript levels of PUMA-G were high in islet beta cells as well as in white adipocytes and spleenocytes from mice. Furthermore, we found low transcript levels of PUMA-G in pancreatic acini and murine beta cell lines, NIT-1 and Min-6.In order to study the relationship between PUMA-G signaling pathway and insulin secretion, we determined the levels of two important second messengers, Ca²⁺ and cAMP in murine islets in the presence of nicotinic acid. We found that cAMP accumulation, instead of calcium transient was inhibited by nicotinic acid in the presence of IBMX or forskolin. As a result, glucose stimulated insulin secretion from murine islets was also inhibited. In contrast, pretreatment with pertussis toxin (PTX) for 24 hrs reduced the inhibitory effect of nicotinic acid. Furthermore, the inhibition was dose dependent. Thus, nicotinic acid inhibited insulin secretion through its stimulation of PUMA-G on islet beta cells.To study the function of PUMA-G in murine islet beta cells directly, we cloned PUMA-G into plasmid pEGFP-N1. The construct was transfected into murine beta cell line NIT-1 through cationic Lipofectamine 2000 (Invitrogen) or electroporation. We found green fluorescence on the membrane of NIT-1 cells transfected with the PUMA-G construct, while the green fluorescence remained in the cytoplasm when pEGFP-N1 was used. Thus, the result demonstrated the membrane location of PUMA-G on NIT-1. Future study will be carried out on this PUMA-G transfectant to confirm the inhibitory effect of nicotinic acid on insulin secretion.3-hydroxybutyrate, which is the predominating ketone body, has been shown to be the cognate ligand for PUMA-G. The concentration of ketone body increases dramatically in the blood of diabetic patients and in animal models like partial pancreatectomy. Therefore, we measured PUMA-G transcript levels before and after 70% pancreatectomy in Balb/c mice. We found that transcript levels of PUMA-G increased significantly within 1-4 days after the surgery as compared to the control, suggesting PUMA-G expression might be important for the regeneration of islet in the pancreas.